Gliomas account for about 60% of all primary CNS tumors. Glioblastoma multiforme (GBM) or grade IV gliomas which comprise two thirds of all gliomas are the most malignant form. Limitation to brain tumor therapy in general is the difficulty in delivering therapeutic agents across the blood-brain barrier (BBB) sufficient to achieve high concentrations at the tumor site. In this project, we will focus on developing a gene delivery system that can change the surface of a tumor cell so that it presents a molecular "beacon" (biotinylated receptors) to attract diagnostic and therapeutic agents to it. This strategy is designed to make tumor cells a selective, "easy target" for different biotinylated-imaging agents (for diagnostics) or biotinylated-diphtheria toxin (for therapy) complexed to streptavidin which binds with extremely high affinity to biotin (Kd = 10-15) and can be internalized into tumor cells. A critical component of this strategy will be to facilitate delivery of these biotinylated therapeutic/diagnostic agents from the vasculature into tumors in the brain using a complex between streptavidin and transferrin. This universal transport vehicle will take advantage of transferrin receptors on blood vessels in the brain which normally facilitate passage of molecules across the blood-brain barrier. This should enhance the delivery of targeted toxins to the brain and therefore increase their therapeutic efficacy for brain tumors by virtue of high affinity binding between biotin on the tumor cell surface and streptavidintoxin complex. The toxin complex will be safe to normal cells which will not take it up by endocytosis, but will be activated in tumor cells by endocytosis mediated by the biotinylated receptors. The advantage of this biotinylation approach is that it allows the imaging of brain tumors with different modalities including Magnetic Resonance (MR), Positron Emission Tomography (PET) as well as Single Photon Emission Computed Tomography (SPECT). The strategies proposed here are designed to have a major impact on the development of more efficient brain tumor therapeutic and imaging systems and can be combined with traditional therapies. The versatility of this technique can be extended to allow the tracking of any cell transduced to express biotin on its surface, including stem cells, and is fully compatible with translation into humans since all components of this system including the biotin-streptavidin system, the different imaging modalities, diphtheria toxin as well as gene transfer have already been used separately in clinical trials in the USA.